Diffusion in heterogeneous systems studied by laser scanning confocal microscopy and fluorescence correlation spectroscopy

Understanding and controlling the mechanism of the diffusion of small molecules, macromolecules and nanoparticles in heterogeneous environments is of paramount fundamental and technological importance. The aim of the thesis is to show, how by studying the tracer diffusion in complex systems, one can obtain information about the tracer itself, and the system where the tracer is diffusing. rnIn the first part of my thesis I will introduce the Fluorescence Correlation Spectroscopy (FCS) which is a powerful tool to investigate the diffusion of fluorescent species in various environments. By using the main advantage of FCS namely the very small probing volume (<1µm3) I was able to track the kinetics of phase separation in polymer blends at late stages by looking on the molecular tracer diffusion in individual domains of the heterogeneous structure of the blend. The phase separation process at intermediate stages was monitored with laser scanning confocal microscopy (LSCM) in real time providing images of droplet coalescence and growth. rnIn a further project described in my thesis I will show that even when the length scale of the heterogeneities becomes smaller than the FCS probing volume one can still obtain important microscopic information by studying small tracer diffusion. To do so, I will introduce a system of star shaped polymer solutions and will demonstrate that the mobility of small molecular tracers on microscopic level is nearly not affected by the transition of the polymer system to a “glassy” macroscopic state. rnIn the last part of the thesis I will introduce and describe a new stimuli responsive system which I have developed, that combines two levels of nanoporosity. The system is based on poly-N-isopropylacrylamide (PNIPAM) and silica inverse opals (iOpals), and allows controlling the diffusion of tracer molecules. rn

Mechanical properties and structure of gel systems

Understanding the origins of the mechanical properties and its correlation withrnthe microstructure of gel systems is of great scientific and industrial interest. Inrngeneral, colloidal gels can be classified into chemical and physical gels, accordingrnto the life time of the network bonds. The characteristic di↵erences in gelationrndynamics can be observed with rheological measurements.rnAs a model system, a mixture of sodium silicate and low concentration sulfuric acidrnwas used. Nano-sized silica particles grow and aggregate to a system-spanning gelrnnetwork. The influence of the finite solubility of silica at high pH on the gelationrnwas studied with classical and piezo rheometer. The storage modulus of therngel grew logarithmically with time with two distinct growth laws. A relaxationrnat low frequency was observed in the frequency dependent measurements. I attributernthese two behaviors as a sign of structural rearrangements due to the finiternsolubility of silica at high pH. The reaction equilibrium between formation andrndissolution of bonds leads to a finite life time of the bonds and behavior similar tornphysical gel. The frequency dependence was more pronounced for lower water concentrations,rnhigher temperatures and shorter reaction times. With two relaxationrnmodels, I deduced characteristic relaxation times from the experimental data. Besidesrnrheology, the evolution of silica gels at high pH on di↵erent length scales wasrnstudied by NMR and dynamic light scattering. The results revealed that the primaryrnparticles existed already in sodium silicate and aggregated after the mixingrnof reactants due to a chemical reaction. Throughout the aggregation process thernsystem was in its chemical reaction equilibrium. Applying large oscillatory shearrnstrain to the gel allowed for modifying the gel modulus. The e↵ect of shear andrnshear history on the rheological properties of the gel were investigated. The storagernmodulus of the final gel increased with increasing strain. This behavior can be explained with (i) shear-induced aggregate compaction and (ii) combination ofrnbreakage and new formation of bonds.rnIn comparison with the physical gel-like behavior of the silica gel at high pH, typicalrnchemical gel features were exhibited by other gels formed from various chemicalrnreactions. Influences of the chemical structure modification on the gelation wererninvestigated with the piezo-rheometer. The external stimuli can be applied to tunernthe mechanical properties of the gel systems.

Monitoring kinetic processes in polymeric colloids by fluorescence correlation spectroscopy

Polymerbasierte Kolloide mit Groen im Nanometerbereich werden als aussichts- reiche Kandidaten fur die Verkapselung und den Transport von pharmazeutischen Wirkstoen angesehen. Daher ist es wichtig die physikalischen Prozesse, die die Bil- dung, Struktur und kinetische Stabilitat der polymerbasierten Kolloide beein ussen, besser zu verstehen. Allerdings ist die Untersuchung dieser Prozesse fur nanome- tergroe Objekte kompliziert und erfordert fortgeschrittene Techniken. In dieser Arbeit beschreibe ich Untersuchungen, bei denen Zwei-Farben-Fluoreszenzkreuz- korrelationsspektroskopie (DC FCCS) genutzt wurde, um Informationen uber die Wechselwirkung und den Austausch von dispergierten, nanometergroen Kolloiden zu bekommen. Zunachst habe ich den Prozess der Polymernanopartikelherstellung aus Emul- sionstropfen untersucht, welcher einen der am haugsten angewendeten Prozesse der Nanopartikelformulierung darstellt. Ich konnte zeigen, dass mit DC FCCS eindeutig und direkt Koaleszenz zwischen Emulsionstropfen gemessen werden kann. Dies ist von Interesse, da Koaleszenz als Hauptgrund fur die breite Groenverteilung der nalen Nanopartikel angesehen wird. Weiterhin habe ich den Austausch von Mizellen bildenden Molekulen zwischen amphiphilen Diblock Kopolymermizellen untersucht. Als Modellsystem diente ein Linear-Burste Block Kopolymer, welches Mizellen mit einer dichten und kurzen Korona bildet. Mit Hilfe von DC FCCS konnte der Austausch in verschiedenen Losungsmitteln und bei verschiedenen Temperaturen beobachtet werden. Ich habe herausgefunden, dass in Abhangigkeit der Qualitat des Losungsmittels die Zeit des Austausches um Groenordnungen verschoben werden kann, was eine weitreichende Einstellung der Austauschkinetik ermoglicht. Eine Eigenschaft die all diese Kolloide gemeinsam haben ist ihre Polydispersitat. Im letzten Teil meiner Arbeit habe ich am Beispiel von Polymeren als Modellsystem untersucht, welchen Eekt Polydispersitat und die Art der Fluoreszenzmarkierung auf FCS Experimente haben. Eine Anpassung des klassischen FCS Modells kann die FCS Korrelationskurven dieser Systeme beschreiben. Die Richtigkeit meines Ansatzes habe ich mit dem Vergleich zur Gel-Permeations-Chromatographie und Brownschen Molekulardynamiksimulationen bestatigt.

Set up of a procedure for correlation analysis between current discharge and dose delivery in a pulsed power plasma device for medical application

This thesis work aims to find a procedure for isolating specific features of the current signal from a plasma focus for medical applications. The structure of the current signal inside a plasma focus is exclusive of this class of machines and a specific analysis procedure has to be developed. The hope is to find one or more features that shows a correlation with the dose erogated. The study of the correlation between the current discharge signal and the dose delivered by a plasma focus could be of some importance not only for the practical application of dose prediction but also for expanding the knowledge anbout the plasma focus physics. Vatious classes of time-frequency analysis tecniques are implemented in order to solve the problem.

Conformationally Controlled Electron Delocalization in n-Type Rods: Synthesis, Structure, and Optical, Electrochemical, and Spectroelectrochemical Properties of Dicyanocyclophanes

A series of dicyanobiphenyl-cyclophanes 1-6 with various pi-backbone conformations and characteristic n-type semiconductor properties is presented. Their synthesis, optical, structural, electrochemical, spectroelectrochemical, and packing properties are investigated. The X-ray crystal structures of all n-type rods allow the systematic correlation of structural features with physical properties. In addition, the results are supported by quantum mechanical calculations based on density functional theory. A two-step reduction process is observed for all n-type rods, in which the first step is reversible. The potential gap between the reduction processes depends linearly on the cos(2) value of the torsion angle phi between the pi-systems. Similarly, optical absorption spectroscopy shows that the vertical excitation energy of the conjugation band correlates with the cos(2) value of the torsion angle phi. These correlations demonstrate that the fixed intramolecular torsion angle phi is the dominant factor determining the extent of electron delocalization in these model compounds, and that the angle phi measured in the solid-state structure is a good proxy for the molecular conformation in solution. Spectroelectrochemical investigations demonstrate that conformational rigidity is maintained even in the radical anion form. In particular, the absorption bands corresponding to the SOMO-LUMO+i transitions are shifted bathochromically, whereas the absorption bands corresponding to the HOMO-SOMO transition are shifted hypsochromically with increasing torsion angle phi.

Ecotypes and genetic structure of Rhinanthus alectorolophus (Orobanchaceae) in southwestern Germany

European annual species of the genus Rhinanthus often exhibit seasonal ecotypic variation, a phenomenon also known from related genera of hemiparasitic Orobanchaceae. Populations with different flowering times exist, correlated with differences in a number of morphological characters. The present study evaluates the correlation of morphological characters and genetic differentiation of populations of Rhinanthus alectorolophus. Thirty-nine populations of three different subspecies from southwestern Germany were sampled. A total of 798 individuals were used for morphological analyses and 187 of these for AFLP analyses. Principal component analysis showed that morphological variation is mostly continuous. In a discriminant analysis based on morphological characters, only 89.7 % of all individuals were correctly assigned to their previously determined subspecies, indicating that subspecies identification is ambiguous for some populations. Using AFLP data and Bayesian assignment analysis, the sampled individuals could be grouped in three genetic clusters which do not correspond to the three subspecies. Instead, the clustering shows a clear geographic pattern and a Mantel test likewise revealed a significant correlation between genetic and geographic distances. Correlations of genetic distances with differences in morphological characters were weak and mostly insignificant. The results indicate that the subspecies of R. alectorolophus do not form discrete entities and that the character combinations distinguishing them are homoplastic.

Correlation between broiler lameness and anatomical measurements of bone using radiographical projections with assessments of consistency across and within radiographs.

Lameness represents a major welfare and production issue in the poultry industry with a recent survey estimating 27% of birds lame and 3% unable to walk by 40 d of age. A variety of factors may induce lameness and are typically grouped into 2 broad classes on the basis of being infectious or skeletal in nature with the latter accounting for the majority of cases. The current work sought to build upon a large body of literature assessing the anatomical properties of bone in lame birds. Our specific objectives sought to identify relationships between relevant anatomical properties of the tibia and metatarsus using digital quantification from radiographs of legs and a measure of walking difficulty. Resulting output was statistically analyzed to assess 1) observer reliability for consistency in placing the leg during the radiograph procedure and quantification of the various measures within a radiograph, 2) the relationship between the various measurements of anatomical bone properties and sex, bird mass, and gait score, and 3) the relationship between each measurement and leg symmetry. Our anatomical bone measures were found to be reliable (intra-rater and test-retest reliabilities < 0.75) within radiograph for all measures and 8 of the 10 measures across radiographs. Several measures of bone properties in the tibia correlated to difficulty walking as measured by gait score (P < 0.05), indicating greater angulations with increasing lameness. Of the measures that manifested a gait score × bird mass interaction, heavier birds appeared to exhibit less angulation with increasing difficulty walking with lighter birds the opposite. These interactions suggest possibilities for influencing effects of activity or feed intake on bone mineralization with the bone angulation observed. Our efforts agree with that of others and indicate that angulation of the tibia may be related to lameness, though subsequent efforts involving comprehensive measures of bird activity, growth rates, and internal bone structure will be needed if the validity of the measures are to be accepted.

Quantitative 3D strain analysis in analogue experiments: Integration of X-ray computed tomography and digital volume correlation techniques

The combination of scaled analogue experiments, material mechanics, X-ray computed tomography (XRCT) and Digital Volume Correlation techniques (DVC) is a powerful new tool not only to examine the 3 dimensional structure and kinematic evolution of complex deformation structures in scaled analogue experiments, but also to fully quantify their spatial strain distribution and complete strain history. Digital image correlation (DIC) is an important advance in quantitative physical modelling and helps to understand non-linear deformation processes. Optical non-intrusive (DIC) techniques enable the quantification of localised and distributed deformation in analogue experiments based either on images taken through transparent sidewalls (2D DIC) or on surface views (3D DIC). X-ray computed tomography (XRCT) analysis permits the non-destructive visualisation of the internal structure and kinematic evolution of scaled analogue experiments simulating tectonic evolution of complex geological structures. The combination of XRCT sectional image data of analogue experiments with 2D DIC only allows quantification of 2D displacement and strain components in section direction. This completely omits the potential of CT experiments for full 3D strain analysis of complex, non-cylindrical deformation structures. In this study, we apply digital volume correlation (DVC) techniques on XRCT scan data of “solid” analogue experiments to fully quantify the internal displacement and strain in 3 dimensions over time. Our first results indicate that the application of DVC techniques on XRCT volume data can successfully be used to quantify the 3D spatial and temporal strain patterns inside analogue experiments. We demonstrate the potential of combining DVC techniques and XRCT volume imaging for 3D strain analysis of a contractional experiment simulating the development of a non-cylindrical pop-up structure. Furthermore, we discuss various options for optimisation of granular materials, pattern generation, and data acquisition for increased resolution and accuracy of the strain results. Three-dimensional strain analysis of analogue models is of particular interest for geological and seismic interpretations of complex, non-cylindrical geological structures. The volume strain data enable the analysis of the large-scale and small-scale strain history of geological structures.

DEFINITION OF THE LANDSCAPE OF CHROMATIN STRUCTURE AT THE FRATAXIN GENE IN FRIEDREICH’S ATAXIA

Friedreich’s ataxia (FRDA) is caused by the transcriptional silencing of the frataxin (FXN) gene. FRDA patients have expansion of GAA repeats in intron 1 of the FXN gene in both alleles. A number of studies demonstrated that specific histone deacetylase inhibitors (HDACi) affect either histone modifications at the FXN gene or FXN expression in FRDA cells, indicating that the hyperexpanded GAA repeat may facilitate heterochromatin formation. However, the correlation between chromatin structure and transcription at the FXN gene is currently limited due to a lack of more detailed analysis. Therefore, I analyzed the effects of the hyperexpanded GAA repeats on transcription status and chromatin structure using lymphoid cell lines derived from FRDA patients. Using chromatin immunoprecipitation and quantitative PCR, I observed significant changes in the landscape of histone modifications in the vicinity of the GAA tract in FRDA cells relative to control cells. Similar epigenetic changes were observed in GFP reporter construct containing 560 GAA repeats. Further, I detected similar levels of FXN pre-mRNA at a region upstream of hyperexpanded GAA repeats in FRDA and control cells, indicating similar efficiency of transcription initiation in FRDA cells. I also showed that histone modifications associated with hyperexpanded GAA repeats are independent of transcription progression using the GFP reporter system. My data strongly support evidence that FXN deficiency in FRDA patients is consequence of defective transition from initiation to elongation of FXN transcription due to heterochromatin-like structures formed in the proximity of the hyperexpanded GAAs.

Structure-function relation in retinoid regulated gene expression

Retinoids are known to inhibit proliferation of and induce terminal differentiation of many normal and transformed cells. It has been postulated that retinoids exert their effect by altering gene expression. HL-60 cells and macrophages both respond to retinoic acid action by the rapid induction of the enzyme tissue transglutaminase. The induction has been shown to be due to increased transcription of the transglutaminase gene. The first part of the dissertation studied the structure-function relationship of retinoid-regulated transglutaminase induction, differentiation and proliferation in HL-60 cells using retinoid analogs. The results indicated strict structural constraints and a strong structure-function correlation between transglutaminase induction and differentiation; those retinoids that induced transglutaminase also induced differentiation, those analogs that did not induce transglutaminase could not induce differentiation. The ability of the retinoids to induce transglutaminase in HL-60 cells was paralleled in macrophages. However, the antiproliferative effect of the retinoids displayed less stringent structural constraints than their differentiation- and transglutaminase-inducing properties. Specifically all the retinoids were able to inhibit proliferation to varying extents. It is concluded that the induction of transglutaminase and of differentiation by retinoids is mediated by receptors. While receptor mediation cannot be entirely ruled out, with the current data no definitive statement can be made about the antiproliferative activity of retinoids. Also, the concordance in the ability of the retinoids to induce transglutaminase and the ability to induce differentiation of HL-60 cells suggests that the former is an early response of the cells to retinoids and differentiation a later consequence on the same pathway. Using the induction of transglutaminase as an index of the direct, or primary, effect of retinoids on gene expression, the second part of the dissertation investigates, by 2D gel electrophoresis, the alteration in the rates of synthesis of other proteins in macrophages and HL-60 cells in response to short incubations with retinoic acid. Any changes in parallel with transglutaminase were taken to indicate proteins directly under the control of retinoic acid. It is concluded that retinoic acid regulates the expression of a circumscribed set of genes in a cell-specific manner. The results support the hypothesis that retinoids exert their multiple effects on myeloid cells, in part, by receptor-mediated alternations in gene expression. ^

Structure/affinity studies in the bicyclo-DNA series: Synthesis and properties of oligonucleotides containing bcen-T and iso-tricyclo-T nucleosides

We present the synthesis of the two novel nucleosides iso-tc-T and bcen-T, belonging to the bicyclo-/tricyclo-DNA molecular platform. In both modifications the torsion around C6’–C7’ within the carbocyclic ring is planarized by either the presence of a C6’–C7’ double bond or a cyclopropane ring. Structural analysis of these two nucleosides by X-ray analysis reveals a clear preference of torsion angle γ for the gauche orientation with the furanose ring in a near perfect 2’-endo conformation. Both modifications were incorporated into oligodeoxynucleotides and their thermal melting behavior with DNA and RNA as complements was assessed. We found that the iso-tc-T modification was significantly more destabilizing in duplex formation compared to the bcen-T modification. In addition, duplexes with complementary RNA were less stable as compared to duplexes with DNA as complement. A structure/affinity analysis, including the already known bc-T and tc-T modifications, does not lead to a clear correlation of the orientation of torsion angle γ with DNA or RNA affinity. There is, however, some correlation between furanose conformation (N- or S-type) and affinity in the sense that a preference for a 3’-endo like conformation is associated with a preference for RNA as complement. As a general rule it appears that Tm data of single modifications with nucleosides of the bicyclo-/tricyclo-DNA platform within deoxyoligonucleotides are not predictive for the stability of fully modified oligonucleotides.

Elucidating the internal structure of psychophysical timing performance in the sub-second and second range by utilizing confirmatory factor analysis

The most influential theoretical account in time psychophysics assumes the existence of a unitary internal clock based on neural counting. The distinct timing hypothesis, on the other hand, suggests an automatic timing mechanism for processing of durations in the sub-second range and a cognitively controlled timing mechanism for processing of durations in the range of seconds. Although several psychophysical approaches can be applied for identifying the internal structure of interval timing in the second and sub-second range, the existing data provide a puzzling picture of rather inconsistent results. In the present chapter, we introduce confirmatory factor analysis (CFA) to further elucidate the internal structure of interval timing performance in the sub-second and second range. More specifically, we investigated whether CFA would rather support the notion of a unitary timing mechanism or of distinct timing mechanisms underlying interval timing in the sub-second and second range, respectively. The assumption of two distinct timing mechanisms which are completely independent of each other was not supported by our data. The model assuming a unitary timing mechanism underlying interval timing in both the sub-second and second range fitted the empirical data much better. Eventually, we also tested a third model assuming two distinct, but functionally related mechanisms. The correlation between the two latent variables representing the hypothesized timing mechanisms was rather high and comparison of fit indices indicated that the assumption of two associated timing mechanisms described the observed data better than only one latent variable. Models are discussed in the light of the existing psychophysical and neurophysiological data.

Altered structure of cortical sulci in gilles de la Tourette syndrome: Further support for abnormal brain development.

Gilles de la Tourette syndrome is a neurodevelopmental disorder characterized by the presence of motor and vocal tics. We hypothesized that patients with this syndrome would present an aberrant pattern of cortical formation, which could potentially reflect global alterations of brain development. Using 3 Tesla structural neuroimaging, we compared sulcal depth, opening, and length and thickness of sulcal gray matter in 52 adult patients and 52 matched controls. Cortical sulci were automatically reconstructed and identified over the whole brain, using BrainVisa software. We focused on frontal, parietal, and temporal cortical regions, in which abnormal structure and functional activity were identified in previous neuroimaging studies. Partial correlation analysis with age, sex, and treatment as covariables of noninterest was performed amongst relevant clinical and neuroimaging variables in patients. Patients with Gilles de la Tourette syndrome showed lower depth and reduced thickness of gray matter in the pre- and post-central as well as superior, inferior, and internal frontal sulci. In patients with associated obsessive-compulsive disorder, additional structural changes were found in temporal, insular, and olfactory sulci. Crucially, severity of tics and of obsessive-compulsive disorder measured by Yale Global Tic severity scale and Yale-Brown Obsessive-Compulsive scale, respectively, correlated with structural sulcal changes in sensorimotor, temporal, dorsolateral prefrontal, and middle cingulate cortical areas. Patients with Gilles de la Tourette syndrome displayed an abnormal structural pattern of cortical sulci, which correlated with severity of clinical symptoms. Our results provide further evidence of abnormal brain development in GTS. © 2015 International Parkinson and Movement Disorder Society.

Preference Structure and Random Paths to Stability in Matching Markets

This paper examines how preference correlation and intercorrelation combine to influence the length of a decentralized matching market's path to stability. In simulated experiments, marriage markets with various preference specifications begin at an arbitrary matching of couples and proceed toward stability via the random mechanism proposed by Roth and Vande Vate (1990). The results of these experiments reveal that fundamental preference characteristics are critical in predicting how long the market will take to reach a stable matching. In particular, intercorrelation and correlation are shown to have an exponential impact on the number of blocking pairs that must be randomly satisfied before stability is attained. The magnitude of the impact is dramatically different, however, depending on whether preferences are positively or negatively intercorrelated.

Bayesian models for the analysis of genetic structure when populations are correlated

Motivation: Population allele frequencies are correlated when populations have a shared history or when they exchange genes. Unfortunately, most models for allele frequency and inference about population structure ignore this correlation. Recent analytical results show that among populations, correlations can be very high, which could affect estimates of population genetic structure. In this study, we propose a mixture beta model to characterize the allele frequency distribution among populations. This formulation incorporates the correlation among populations as well as extending the model to data with different clusters of populations. Results: Using simulated data, we show that in general, the mixture model provides a good approximation of the among-population allele frequency distribution and a good estimate of correlation among populations. Results from fitting the mixture model to a dataset of genotypes at 377 autosomal microsatellite loci from human populations indicate high correlation among populations, which may not be appropriate to neglect. Traditional measures of population structure tend to over-estimate the amount of genetic differentiation when correlation is neglected. Inference is performed in a Bayesian framework.